The invention herein described is an improvement in or modification of the invention described in our co-pending application PCT/AU96/00365, the contents of which are incorporated herein by reference.
The invention will herein be described with particular reference to a biosensor adapted to measure the concentration of glucose in blood, but it will be understood not to be limited to that particular use and is applicable to other analytic determinations.
It is known to measure the concentration of a component to be analysed in an aqueous liquid sample by placing the sample into a reaction zone in an electrochemical cell comprising two electrodes having an impedance which renders them suitable for amperometric measurement. The component to be analysed is allowed to react directly with an electrode, or directly or indirectly with a redox reagent whereby to form an oxidisable (or reducible) substance in an amount corresponding to the concentration of the component to be analysed. The quantity of the oxidisable (or reducible) substance present is then estimated electrochemically. Generally this method requires sufficient separation of the electrodes so that electrolysis products at one electrode cannot reach the other electrode and interfere with the processes at the other electrode during the period of measurement.
In our co-pending application we described a novel method for determining the concentration of the reduced (or oxidised) form of a redox species in an electrochemical cell of the kind comprising a working electrode and a counter (or counter/reference) electrode spaced from the working electrode. The method involves applying an electrical potential difference between the electrodes, spacing the working electrode from the counter electrode so that reaction products from the counter electrode arrive at the working electrode and selecting the potential of the working electrode so that the rate of electro-oxidation of the reduced form of the species (or of electro-reduction of the oxidised form) is diffusion controlled. By determining the current as a function of time after application of the potential and prior to achievement of a steady state current and then estimating the magnitude of the steady state current, the method previously described allows the diffusion coefficient and/or the concentration of the reduced (or oxidised) form of the species to be estimated.
Our co-pending application exemplifies this method with reference to use of a "thin layer" cell employing a GOD/Ferrocyanide system. As herein used, the term "thin layer electrochemical cell" refers to a cell having closely spaced electrodes such that reaction products from the counter electrode arrive at the working electrode. In practice, the separation of electrodes in such a cell for measuring glucose in blood will be less than 500 microns, and preferably less than 200 microns.
The chemistry used in the exemplified electrochemical cell is as follows: ##STR1## where GOD is the enzyme glucose oxidase, and GOD* is the `activated` enzyme. Ferricyanide (Fe(CN).sub.6 !.sup.3-) is the `mediator` which returns the GOD* to its catalytic state. GOD, an enzyme catalyst, is not consumed during the reaction so long as excess mediator is present. Ferrocyanide (Fe(CN).sub.6 !.sup.4-) is the product of the total reaction. Ideally there is initially no ferrocyanide, although in practice there is often a small quantity. After reaction is complete the concentration of ferrocyanide (measured electrochemically) indicates the initial concentration of glucose. The total reaction is the sum of reactions 1 and 2: ##STR2##
The prior art suffers from a number of disadvantages. Firstly, sample size required is greater than desirable. It would be generally preferable to be able to make measurements on samples of reduced volume since this in turn enables use of less invasive methods to obtain samples.
Secondly, it would be generally desirable to improve the accuracy of measurement and to eliminate or reduce variations due, for example, to cell asymmetry or other factors introduced during mass production of microcells.
Thirdly, it would be generally desirable to reduce the time that is required in which to obtain a measurement. The test protocols used in current commercially available electrochemical glucose sensors involve a predetermined wait period at the beginning of the test during which the enzyme reacts with the glucose to produce the specie that is sensed electrochemically. This initial period is fixed at the maximum necessary to achieve the desired reaction under all conditions of use.
Fourthly, it would be desirable to eliminate variations due to oxygen. Oxygen can be plentiful in blood, either dissolved in the plasma, or bound in hemoglobin. It can also be introduced during "finger sticking", where a blood drop of small volume and high surface area is exposed to the atmosphere prior to introduction to a cell. Oxygen can interfere since oxygen is a mediator for GOD. The reaction is as follows: ##STR3##
In most situations the complication of oxygen also acting as a mediator is unwanted, simply because the concentration of final ferrocyanide no longer is directly proportional to the concentration of initial glucose. Instead, the initial glucose concentration is then related to both the final concentration of ferrocyanide and of hydrogen peroxide.